Fruiting Body Formation Research Articles

The genome sequence of the commercially cultivated mushroom Agrocybe aegerita reveals a conserved repertoire of fruiting-related genes and a versatile suite of biopolymer-degrading enzymes

BackgroundAgrocybe aegerita is an agaricomycete fungus with typical mushroom features, which is commercially cultivated for its culinary use. In nature, it is a saprotrophic or facultative pathogenic fungus causing a white-rot of hardwood in forests of warm and mild climate. The ease of cultivation and fructification on solidified media as well as its archetypal mushroom fruit body morphology render A. aegerita a well-suited model for investigating mushroom developmental biology.ResultsHere, the genome of the species is reported and analysed with respect to carbohydrate active genes and genes known to play a role during fruit body formation. In terms of fruit body development, our analyses revealed a conserved repertoire of fruiting-related genes, which corresponds well to the archetypal fruit body morphology of this mushroom. For some genes involved in fruit body formation, paralogisation was observed, but not all fruit body maturation-associated genes known from other agaricomycetes seem to be conserved in the genome sequence of A. aegerita. In terms of lytic enzymes, our analyses suggest a versatile arsenal of biopolymer-degrading enzymes that likely account for the flexible life style of this species. Regarding the amount of genes encoding CAZymes relevant for lignin degradation, A. aegerita shows more similarity to white-rot fungi than to litter decomposers, including 18 genes coding for unspecific peroxygenases and three dye-decolourising peroxidase genes expanding its lignocellulolytic machinery.ConclusionsThe genome resource will be useful for developing strategies towards genetic manipulation of A. aegerita, which will subsequently allow functional genetics approaches to elucidate fundamentals of fruiting and vegetative growth including lignocellulolysis.

Functional analysis of an APSES transcription factor (GlSwi6) involved in fungal growth, fruiting body development and ganoderic-acid biosynthesis in Ganoderma lucidum

The APSES transcription factors have been identified as key regulators of fungal development and other biological processes in fungi. In the present study, the function of Ganoderma lucidum GlSwi6, a homolog of Saccharomyces cerevisiae Swi6, was characterized. RNAi was used to examine the function of GlSwi6 in G. lucidum. Silencing GlSwi6 resulted in multiple developmental defects, including reduced fungal growth and increased hyphal branching, and the GlSwi6-silenced strains did not exhibit primordium or fruiting body formation. In addition, the H2O2 and ganoderic-acid (GA) levels of the GlSwi6-silenced strains decreased approximately 50% and 25%, respectively, compared with those of the WT strain. Furthermore, the addition of H2O2 led to the recovery of the GA levels of GlSwi6-silenced strains, implying that GlSwi6 might regulate GA biosynthesis by regulating the intracellular ROS levels. Taken together, these results indicate that GlSwi6 is involved in fungal growth, development and GA biosynthesis in G. lucidum.

Influencia de la luz azul sobre la productividad del cultivo sólido de Ganoderma lucidum

Ganoderma lucidum es un hongo macromiceto reconocido por sus propiedades medicinales y su contenido de compuestos bioactivos que incluyen polisacáridos, triterpenoides, proteínas inmunomoduladoras, entre otros, lo que ha generado un incremento notable en su producción. La mayoría de especies de hongos responden y se adaptan a diversas señales ambientales incluida la luz, que favorece su productividad, tanto en calidad como en cantidad al estar estrechamente relacionada con la formación de cuerpos fructíferos. Por tal razón, el objetivo de este estudio fue evaluar la eficiencia biológica (EB) y la tasa de producción (TP) como parámetros de productividad del cultivo sólido de Ganoderma lucidum bajo irradiación de los sustratos con luz emitida por diodos azules (LED) con dos periodos de foto-estímulo de 12 y 24 h durante todas las fases de cultivo para inducir el crecimiento micelial y la formación de los cuerpos fructíferos. Se aplicaron parámetros convencionales para el crecimiento y desarrollo del hongo en las etapas de producción. Para la formulación de los sustratos, se emplearon residuos agroindustriales y materiales lignocelulósicos. El diámetro de los cuerpos fructíferos sometidos a tratamientos con luz azul fue mayor que los exhibidos a luz blanca fluorescente (Testigo). Los resultados muestran que el cultivo de Ganoderma lucidum con exposición a la luz azul es útil para la inducción de cuerpos fructíferos de alta calidad, logrando una disminución del periodo de fermentación en 16 días para el foto-estímulo de 24 h con EB de 28,04% y TP de 0,64.

Analysis of Random Migration of Dictyostelium Amoeba in Confined and Unconfined Environments.

Dictyostelium discoideum has proven to be an excellent model to study amoeboid cell migration. During their life cycle, Dictyostelium cells exhibit distinct modes of motility. Individual growth-phase cells explore new territories by random cell migration using the core cell motility machinery, but they can also hunt bacteria by detection and chemotaxis toward the by-product folate. After depletion of nutrients, the cells initiate a developmental program allowing streaming of the cells into aggregation centers by chemotaxis toward cAMP and by cell-to-cell adhesion. Subsequent development is associated with complex rotational movement of the compacted aggregates to drive cell type specific sorting, which in turn is necessary for terminal culmination and formation of fruiting bodies. Here we describe a protocol for the analyses of cell motility of vegetative Dictyostelium cells in unconfined and mechanically confined settings.

Identification of Optimal Culture Conditions for Mycelial Growth and Cultivation of Monkey Head Mushrooms (Hericium erinaceus (Bull.: fr.) Pers)

Monkey head mushrooms (Hericium erinaceus (Bull.: Fr.) Pers) have been broadly cultivated and widely consumed as traditional medicinal herbs as well as functional food in the Orient for several hundred years of history. The identification of optimal culture conditions for mycelium growth and fruiting body formation is one of the most important steps in cultivation of mushroom. The aim of this study was to investigate the optimal culture conditions including pH level, temperature, media and substrate mixtures for the mycelium growth  and cultivation of Hericium erinaceus strain He-2. Results of the study revealed that the optimal conditions for mycelial growth were observed at 25 ± 1oC and pH 8.0. H. erinaceus was cultured on five different types of culture media: Czapek, Raper, PGA (potato, glucose, agar), PGA supplemented with rice bran, and PGA supplemented with fresh mushrooms. PGA supplemented with fresh mushrooms was found to be the best medium for the growth of mycelia. A media containing 99% grain of rice + 1% CaCO3 was considered as the best mother spawn media for mycelial growth. Among various culture media, the highest mycelium growth rate and biological efficiency of H. erinaceus were obtained when grown on a treatment of 87% sawdust + 4% corn bran + 8% rice bran + 1% CaCO3.

Substrate optimization for cultivation of Pleurotus ostreatus on lignocellulosic wastes (coffee, sawdust, and sugarcane bagasse) in Mizan–Tepi University, Tepi Campus, Tepi Town

Optimization of oyster (Pleurotus ostreatus) mushroom cultivation using locally available substrates and materials in Debre Berhan, EthiopiaAsmamaw Tesfaw, Abebe Tadesse, Gebre Kiros

Phenotypic and Genetic Diversity of the Culinary-Medicinal Winter Mushroom Flammulina velutipes (Agaricomycetes) in China.

Flammulina velutipes is one of the mushrooms produced most widely in East Asia. In this study we investigated phenotypic variations of 7 agronomic traits in 37 F. velutipes strains from China, and we analyzed their genetic diversity with 70 simple sequence repeat markers. The Shannon information index and gene diversity were 0.894 and 0.478, respectively, demonstrating high genetic variation among the tested strains. Poor genetic variation was found among white strains, in contrast to yellow ones. Analysis of population structure resolved 2 unambiguous genetic groups in the tested F. velutipes strains, with little differentiation between them (FST = 0.016). Yellow cultivars possibly originated from indigenous wild strains in southwest China. Phenotypic correlations were identified among the 7 traits. In particular, stipe length (SL) was significantly positively correlated with yield, indicating that SL could be used as an index for breeding high-yield strains. The 2 genetic groups, and white strains and yellow ones, showed significant differences between SL, yield, and the time interval (days) from mycelial scratch to formation of the first fruiting body. These results indicate that these 3 traits were stratified by population structure. Detection of genetic and phenotypic variations would lay the groundwork for further breeding of elite F. velutipes strains.

Influence of relative humidity and temperature on cultivation of Pleurotus species

Fungi exhibit different behavior under different conditions and react to light, temperature, moisture content etc. The objective of this study was to evaluate the degradation capability of three common white rot fungi, namely: Pleurotus ostreatus, P. pulmonarius, and Lentinus sajor-caju. The respective fungi were cultivated on rice straw under three different environmental conditions for 90 days. The fungi were collected, pure cultured, DNA extracted, and sequenced by ITS regions. The highest consumption of substrate occurred under the Cellar (dark) exposure condition with P. pulmonarius producing the least mass loss. The least amount of degradation occurred under the Air (daylight) condition for all of the fungi with the exception of P. pulmonarius. Exposure to light promoted the formation of fruiting bodies.

Study on the Cultivation of <i>Agaricus blazei</i> (Almond Mushroom) Grown on Compost Mixed with Selected Agro-Residues

The Agaricus blazei strain (KUMB 1221) from Forest Production Control Laboratory, Kyushu University was grown on the basal media of compost mixed with selected agro-residues (sawdust, woodchips and corncob) in the ratio of 25%, 50%, 75% and 100% (by weight). The data was collected on the colony diameter of mycelial growth, days required for spawn run (colonization), days required for primordial formation, whiteness measurement, days required for fruit body formation, number, size and fresh weight of mushrooms, biological efficiency (BE) and mushroom production (MP). It was observed that the mycelial growth showed two types of mycelia according the level of mycelial density; compact (C) and somewhat compact (SC). The fastest spawn run (17 days) was found in woodchips (50%) among all the different treatments while the least spawn run took 26 days in corncob (75%). Sawdust substrates promoted longer days for primordial formation and fruit body development, if compared with woodchips substrates and corncob substrates. With regard to yield, 100%, 75% and 50% mixture with compost were superior to 25% mixture with compost in each group of selected agro-residues and it indicated that adding more compost gave the increased yield. Interestingly, it was noted that mushroom size on compost (100%) was double to the mushroom size of other treatments. In conclusion, it was clearly showed that compost (100%), woodchips (25%) and corncob (25%) could produce better yields among all treatments, and it was also possible to obtain acceptable yields of good quality almond mushroom using main substrates of compost mixed with different agro-residues at various concentrations.

Homospermidine Lipids: A Compound Class Specifically Formed during Fruiting Body Formation of Myxococcus xanthus DK1622.

The fascinating ability of myxobacteria to form multicellular spore filled fruiting bodies under starvation conditions was widely studied as a model for cooperative microbial behavior. The potential of a life cycle induced change of secondary metabolism, as a means to discover novel natural products, remains largely underexplored. We therefore studied the model organism Myxococcus xanthus DK1622 under submersed and solid cultivation conditions to find putatively life-cycle related compounds by applying statistical analysis on analytical data. Utilizing the advantageous characteristics of LC-MS, LC-MS/MS, and MALDI-MSI allowed the identification of compounds unambiguously associated with myxobacterial fruiting bodies. Our screening effort resulted in the purification and structure elucidation of a novel compound, the homospermidine lipid, from cultures that had undergone the fruiting process. A combination of molecular networking and targeted LC-MS/MS in conjunction with our in-house metabolomics database subsequently revealed alternative producers of the respective compound as well as a number of compounds belonging to the same structural class. Three further members of this compound class were isolated from an alternative producer and structurally elucidated by NMR. Insights into the biosynthesis of this novel compound class was gained by feeding of isotopically labeled substrates and in silico analysis.

ECOLOGICAL AND GENETIC ADVANCES IN THE CULTIVATION OF TUBER SPP.

Recent advances on the ecology and genetics of true truffles (Tuber spp.) are reported and their impact on truffle cultivation is discussed. New insights have been gained on truffle soil ecology and interrelationships of truffles with associated microorganisms in the soil. For instance, some bacteria seem to play a key role in truffle fruiting body formation and maturation. However, the most important advance in truffle genetics over the past 20 years has been the sequencing of the Tuber melanosporum genome and the discovery that truffles, like other Pezizalean fungi, are heterothallic. This finding has had a significant impact on research on truffles and many studies have been devoted to better understanding the distribution of the mating types in soil in natural and cultivated truffières. The characterization of the mating type idiomorphs of several Tuber species has led to the possibility of selecting mycelial strains for truffle cultivation in particular sites.

A conserved stem of the Myxococcus xanthus sRNA Pxr controls sRNA accumulation and multicellular development

The small RNA (sRNA) Pxr negatively controls multicellular fruiting body formation in the bacterium Myxococcus xanthus, inhibiting the transition from growth to development when nutrients are abundant. Like many other prokaryotic sRNAs, Pxr is predicted to fold into three stem loops (SL1-SL3). SL1 and SL2 are highly conserved across the myxobacteria, whereas SL3 is much more variable. SL1 is necessary for the regulatory function of Pxr but the importance of SL3 in this regard is unknown. To test for cis genetic elements required for Pxr function, we deleted the entire pxr gene from a developmentally defective strain that fails to remove Pxr-mediated blockage of development and reintroduced variably truncated fragments of the pxr region to test for their ability to block development. These truncations demonstrated that SL3 is necessary for Pxr function in the defective strain. We further show that a highly conserved eight-base-pair segment of SL3 is not only necessary for Pxr to block development in the defective strain under starvation conditions, but is also required for Pxr to prevent fruiting body development by a developmentally proficient wild-type strain under high-nutrient conditions. This conserved segment of SL3 is also necessary for detectable levels of Pxr to accumulate, suggesting that this segment either stabilizes Pxr against premature degradation during vegetative growth or positively regulates its transcription.

Complete Genome Sequence of the Fruiting Myxobacterium Melittangium boletus DSM 14713.

ABSTRACTThe formation of spore-filled fruiting bodies in response to starvation represents a hallmark of many members of the order Myxococcales. Here, we present the complete 9.9-Mb genome of the fruiting type strain Melittangium boletus DSM 14713, the first member of this genus to have its genome sequenced.

Genetic barcoding of dark-spored myxomycetes (Amoebozoa)-Identification, evaluation and application of a sequence similarity threshold for species differentiation in NGS studies.

Unicellular, eukaryotic organisms (protists) play a key role in soil food webs as major predators of microorganisms. However, due to the polyphyletic nature of protists, no single universal barcode can be established for this group, and the structure of many protistean communities remains unresolved. Plasmodial slime moulds (Myxogastria or Myxomycetes) stand out among protists by their formation of fruit bodies, which allow for a morphological species concept. By Sanger sequencing of a large collection of morphospecies, this study presents the largest database to date of dark-spored myxomycetes and evaluate a partial 18S SSU gene marker for species annotation. We identify and discuss the use of an intraspecific sequence similarity threshold of 99.1% for species differentiation (OTU picking) in environmental PCR studies (ePCR) and estimate a hidden diversity of putative species, exceeding those of described morphospecies by 99%. When applying the identified threshold to an ePCR data set (including sequences from both NGS and cloning), we find 64 OTUs of which 21.9% had a direct match (>99.1% similarity) to the database and the remaining had on average 90.2±0.8% similarity to their best match, thus thought to represent undiscovered diversity of dark-spored myxomycetes.

Whole-Genome Sequence of the Fruiting Myxobacterium Cystobacter fuscus DSM 52655.

ABSTRACTAmong myxobacteria, the genus Cystobacter is known not only for fruiting body formation but also for formation of secondary metabolites, such as cystobactamids and cystothiazols. Here, we present the complete genome sequence of the Cystobacter fuscus strain DSM 52655, which comprises 12,349,744 bp and 9,836 putative protein-coding sequences.

Draft Genome Sequence of the Fruiting Myxobacterium Nannocystis exedens DSM 71.

ABSTRACTIn response to starvation, members of the order Myxococcales form morphologically very different fruiting bodies. To determine whether fruiting myxobacteria share a common genetic program that leads to fruiting body formation, we sequenced and assembled the genome of Nannocystis exedens DSM 71 as two contigs with a total GC content of 72%.

Permethrin drastically affects the developmental cycle of the non-target slime mould Dictyostelium discoideum

The use of pyrethroids has increased throughout the world over the past few decades, as organophosphate, carbamate and organochlorine insecticides are being phased out. Permethrin is widely used in the USA for crops treatment, at concentrations around 750 × 103 μg/L. In our study 3.6 μg/L permethrin decreases the fission-rate and the fruiting bodies formation of slime mould Dictyostelium discoideum. Whereas 3.6 × 104 μg/L kills the 100% of amoebae, showing a 24 h-LC50 = 96.6 μg/L. This concentration induces an increase in the pseudocholinesterase activity as well as in both butyrylcholinesterase and heat-shock-protein 70 presence. Our results highlight the high sensitivity of Dictyostelium to permethrin, at concentration of about 105 lesser than what used for agricultural pest control. If we match our results on 6 days of exposure, with the permethrin relatively slow permanence (30 days) in the aerobic soil, as well as the higher effect of permethrin than organophosphate, carbamate and organochlorine pesticides on D. discoideum, the damage on the dictyostelids community, by use of permethrin, is clear. Our data suggest that, if the sustainable agriculture implementation is a topic of the modern “industrial” farming, the permethrin cannot represent a reliable alternative to organochlorine, organophosphate or carbamate pesticides, in implementing Integrated Pest Management programmes.

SUB1 has photoreceptor dependent and independent functions in sexual development and secondary metabolism in Trichoderma reesei.

Light dependent processes are involved in the regulation of growth, development and enzyme production in Trichoderma reesei. The photoreceptors BLR1, BLR2 and ENV1 exert crucial functions in these processes. We analyzed the involvement of the transcription factor SUB1 in sexual development as well as secondary metabolism and its position in the light signaling cascade. SUB1 influences growth and in contrast to its homologue in N. crassa, SUB1 is not essential for fruiting body formation and male fertility in T. reesei, but required for female fertility. Accordingly, SUB1 is involved in the regulation of the pheromone system of T. reesei. Female sterility of mutants lacking env1 is rescued in triple mutants of blr1, blr2 and env1, but not in double mutants of these genes. Confrontation of strains lacking sub1 results in growth arrest prior to contact of the potential mating partners. This effect is at least in part due to altered secondary metabolite production. Additionally, together with BLR1 and BLR2, SUB1 is essential for spore pigmentation and transcription of pks4, and secondary metabolism is regulated by SUB1 in a light- and nutrient dependent manner. Our results hence indicate rewiring of several pathways targeted by SUB1 in T. reesei.

The Aspergillus flavus Homeobox Gene, hbx1, is Required for Development and Aflatoxin Production.

Homeobox proteins, a class of well conserved transcription factors, regulate the expression of targeted genes, especially those involved in development. In filamentous fungi, homeobox genes are required for normal conidiogenesis and fruiting body formation. In the present study, we identified eight homeobox (hbx) genes in the aflatoxin-producing ascomycete, Aspergillus flavus, and determined their respective role in growth, conidiation and sclerotial production. Disruption of seven of the eight genes had little to no effect on fungal growth and development. However, disruption of the homeobox gene AFLA_069100, designated as hbx1, in two morphologically different A. flavus strains, CA14 and AF70, resulted in complete loss of production of conidia and sclerotia as well as aflatoxins B1 and B2, cyclopiazonic acid and aflatrem. Microscopic examination showed that the Δhbx1 mutants did not produce conidiophores. The inability of Δhbx1 mutants to produce conidia was related to downregulation of brlA (bristle) and abaA (abacus), regulatory genes for conidiophore development. These mutants also had significant downregulation of the aflatoxin pathway biosynthetic genes aflC, aflD, aflM and the cluster-specific regulatory gene, aflR. Our results demonstrate that hbx1 not only plays a significant role in controlling A. flavus development but is also critical for the production of secondary metabolites, such as aflatoxins.

Complete Genome Sequence of the Fruiting Myxobacterium Myxococcus macrosporus Strain DSM 14697, Generated by PacBio Sequencing

ABSTRACTMembers of the Myxococcales order initiate a developmental program in response to starvation that culminates in formation of spore-filled fruiting bodies. To investigate the genetic basis for fruiting body formation, we present the complete 8.9-Mb genome sequence of Myxococcus macrosporus strain DSM 14697, generated using the PacBio sequencing platform.